1. Field of the Invention
The present invention is drawn to a nucleoside triphosphate (3′-O-FL-dNTP) having a fluorescent 3′-O-blocking group which is chemically removable and a DNA sequencing method (sequencing-by-synthesis) using the same.
2. Description of the Related Art
By virtue of successful result of the Human Genome Project (HGP) recently completed, information of human genome could be obtained. Although this information contains common sequences of the human genome, it does not present individual variation in the genome. To acquire information of complete individual genomes, personal genomes have to be analyzed. Since the haploid human genome occupies approximately 3 billion DNA base pairs, if the capillary electrophoresis-based Sanger method, which has been widely used for DNA sequencing so far, is used for sequencing of a personal genome, the similar cost and time as required for the completion of the HGP will be needed to obtain information of the individual genome (G. M Church et al., Nature Reviews, 2004, 5, 335-344). Since 2004, the National Human Genome Research Institute (NHGRI, USA) has supported the development of technologies enabling whole genome sequencing of each individual with remarkably reduced costs, particularly at a cost of 100,000 dollars in a short term and 1,000 dollars in the long term. As a result, sequencing platforms capable of performing whole gemome sequencing of an individual at a cost of about 50,000 dollars are now commercialized. These machines are based on two major technologies, a pyrosequencing and a sequencing-by-synthesis (SBS). GS TLX Titanium series machine provided by 454 Life Sciences, a Roche Company is based on the pyrosequencing technology. It is possible to analyze 400-600 million bases for 10 hours and to analyze comparatively long chains (approximately 200 nucleotides) at once. However, Genome Analyzer provided by Solexa, Inc. is based on the SBS technology.
The sequencing-by-synthesis (SBS) which is directly related to the present invention, uses fluorescently labeled nucleotides. Particularly, according to the technology, each nucleotide is incorporated into a DNA primer by polymerases and then fluorescent signal from the nucleotide is detected to identify base of the nucleotide and the complementary base can be analyzed at the same time (see FIG. 2).
Nucleoside triphosphates (dNTPs) used for the SBS are basically dual-modified reversible terminators (DRTs), which are modified dNTPs having a reversible blocking group on the 3′-OH moiety (3′-O-blocking group) and a fluorophore on the base (see Figure, left). At this time, 4 bases (A, T, G, and C) are labeled with different fluorophores emitting lights of different wavelengths. A target DNA is used as a template for the polymerization using such modified dNTPs. Then, a nucleotide is once incorporated into a primer chain by DNA polymerases, the other dNTPs cannot be incorporated into the chain because of 3′-O-blocking group of the incorporated nucleotide. Then, the fluorescence of the fluorophore conjugated to the base of the incorporated nucleotide is detected to identify base of the incorporated nucleotide, leading to the analysis of the complementary nucleotide sequence of the template chain. When the fluorophore and the 3′-O-blocking group are removed, a free 3′-OH functional group is recovered, so that the next nucleotide can be incorporated. The base of the incorporated nucleotide can be identified by the same manner as described above, resulting in sequencing the template chain. This step by step sequencing is called ‘sequencing-by-synthesis (SBS)’.
The dual-modified reversible terminators (DRTs) used for commercial SBS has a fluorescent group connected to a base via an acetylene linker. In that case, even though a 3′-O-blocking group and a fluorescent group are removed after DRT insertion and sequencing, the linker part connecting the fluorescent group to the base still remains. This remnant is called a molecular scar. As the sequencing progresses, a newly polymerized chain contains more molecular scars. Since such an accumulation of molecular scars reduces the activity and fidelity of polymerases later on, resulting in limiting the number of sequenced bases. Because of this problem, the SBS is known to have relatively short read-length ca. 25-35 bases, compared with pyrosequencing-based methods. Thus, this limit might increase chances of errors in profiling polynucleotides fragments when a long polynucleotide, which is cut into several short fragments for SBS, is analyzed.
There are two important factors to successfully perform the SBS. First, a polymerase capable of incorporating a DRT with almost perfect efficiency is required. Second, the fluorophore connected to a base and the 3′-O-blocking group have to be removed with almost perfect efficiency in aqueous solution without damaging DNA. Natural DNA polymerases have been evolved for long time so as to receive dNTPs having free 3′-OH selectively. Therefore, they do not accept a substrate when it has a blocking group at 3′-OH. In the earlier study, Sarfati et al. reported in 1994 that dNTPs having 3′-O-ester anthranylic blocking groups were incorporated in a DNA chain by many polymerases (AMV reverse transcriptase, Taq DNA polymerase, and Klenow fragment of DNA polymerase I) and then a free 3′-OH functional group could be recovered by cutting off ester bond using esterase to continue polymerization (Sarfati et al, Gene, 1994, 148: 1-6). In the same year, M. L. Metzker, et al. reported that they could observe the insertion of dNTPs having a 3′-OH group conjugated with different blocking groups by the action of polymerases (AMV reverse transcriptase, M-MuLV reverse transcriptase, Klenow fragment of DNA polymerase I, Sequenase, Bst DNA polymerase, AmpliTaq DNA polymerase, Vent(exo-) DNA polymerase, rTth DNA polymerase, and Pfu(exo-) DNA polymerase) and as a result, dNTPs having a 3′-O-methyl group and a 3′-O-(2-nitrobenzyl) group could be inserted via some of polymerases to terminate polymerization temporarily (M. L. Metzker et al., Nucleic Acids Research, 1994, 22: 4259-4267). However, those prior arts are not suitable to be used in the SBS with respect to efficiency and fidelity for the continuous polymerization.
The reason why dNTPs having a bulky 3′-O-blocking group cannot be accepted as a reaction substrate by polymerases might be explained by narrow space for the 3′-OH group in the active sites of natural polymerases (Burgess et al. A Chemistry European Journal, 1999, 5: 951-960). To generally perform the SBS using dNTPs having a 3′-O-blocking group, a suitably modified polymerase having a larger space surrounding the 3′-OH group in its active site is needed. Recently, professor Ju's group at Columbia University performed the SBS using 3′-O-allylated dNTPs and 3′-O-aziomethylated dNTPs (Seo et al. PNAS, 2005, 102: 5926-5931; Guo et al. PNAS, 2008, 105: 9145-9150). To insert such nucleotides having 3′-O-blcking groups, the above group used a modified DNA polymerase obtained from the strain collected from an Eastern Pacific crater (Southworth et al. PNAS, 1996, 93: 5281-5285). This modified DNA polymerase is now commercially available under the brand name of ‘Therminator II’ provided by New England Biolabs, Inc. Kwiatkowski et al. of Helicos Biosciences Corp. designed 3′-O-hydrocarbyldithiomethyl dNTPs for the SBS (Kwiatkowski et al., 2007, US20077279563), and Parce et al. of Caliper Life Sciences, Inc. used a blocking group containing a phosphate group or a carbamate group (Parce et al., 2006, US20067105300).
If, instead of DRTs, the SBS could utilize mono-modified reversible terminators (MRTs), in which the reversible blocking group on the 3′-OH group played a dual role as a fluorescence-signal reporter as well as a reversible terminator, fluorescent labels on nucleobases would no longer be needed. The MRTs could then be transformed back into the natural state without residual molecular scars after removal of the fluorescent blocking groups from 3′-OH moieties (see FIG. 1, right). However, this principal of sequencing using MRTs and conventional polymerases has not been realized yet.
The present inventors completed the present invention by confirming that the SBS can be successfully carried out by the processes of 1) designing a MRT having a chemical structure playing a role as a blocking group as well as capable of emitting a fluorescence-signal in its 3′-OH group not in its base, and 2) inserting the MRT using conventional polymerases→sequencing by the recognition of fluorescence signal of a 3′-0-fluorescent blocking group→removing the fluorescent blocking group→the second insertion of the MRT (see FIG. 2).